Methods for evaluation of the effectiveness of a drug or drug candidate in treating an inflammatory bowel disease by use of a multiplexed assay kit comprising various biomarkers

ABSTRACT

Disclosed are methods for conducting diagnostic tests for the detection of the inflammatory bowel diseases, such as Crohn&#39;s disease and ulcerative colitis. Also described are methods for monitoring a patient by administering tests of the present invention. Also described are methods for monitoring patient&#39;s treatment by administering tests of the present invention. Also described are methods for evaluating the effectiveness of a drug or a drug candidate by administering tests of the present invention to samples from patients, animal models, and cell cultures treated with a drug or a drug candidate. Also disclosed are methods for determining the usefulness of analytes, e.g. cytokines, for acting as diagnostic and monitoring markers for inflammatory bowel disease in the various methods of the invention.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/716,743, filed, Sep. 27, 2017, which is a continuation of U.S.application Ser. No. 14/478,221, filed Sep. 5, 2014, now U.S. Pat. No.9,804,173, which is a continuation of U.S. application Ser. No.13/775,952, filed Feb. 25, 2013, which is now abandoned, which is adivisional of U.S. application Ser. No. 12/818,093, filed Jun. 17, 2010,which is now abandoned, which is a continuation of U.S. application Ser.No. 11/301,274, filed Dec. 9, 2005, which is now abandoned, and whichclaims priority to U.S. Provisional Application No. 60/634,590, filedDec. 9, 2004, each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This application relates to assay methods, modules, and kits forconducting diagnostic assays for inflammatory diseases.

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD) encompasses a group of diseases such asulcerative colitis (UC) and Crohn's disease (CD). IBDs can be difficultto diagnose. An initial diagnosis, made on the basis of medical historyand physical examination, is generally confirmed via imaging tests tolook at the intestines and laboratory culture tests to rule outbacterial, viral and parasitic infections. The conditions may goundiagnosed for years because symptoms usually develop gradually andless than all of the intestines may be involved.

Colonoscopy can be used to image the intestines and colon. A doctor usesa thin, lighted endoscope to look at the entire intestines anddistinguish between IBDs on the basis of the location of ulcerations.Crohn's disease affects some areas of the intestines and skips overothers. Ulcerative colitis is more indiscriminate. Endoscopy is alsoused to take a biopsy of intestinal tissue, which can be used toidentify the deep inflammation of the bowel that is characteristic ofCrohn's disease. X-rays (after oral or rectal ingestion of Barium),computed tomography (CT) scan, and magnetic resonance imaging (MRI) maybe helpful in locating fistulas.

A stool analysis (including a test for blood in the stool) is oftenperformed, depending on symptoms, to look for blood and signs ofbacterial infection. Blood and urine tests may be done to check foranemia, high white cell counts, or malnutrition-all signs of IBDs.

Currently there is no reliable biochemical test available for IBD.Up-regulation of certain cytokines has been detected in tissue andmucosal samples surgically removed from diseased bowel in IBD patients(Indaram A. V. et al., Mucosal cytokine production in radiation-inducedproctosigmoiditis compared with inflammatory bowel disease, Am J.Gastroenterol. 2000 95(5): 1221-5; McCormack G, et al., Tissue cytokineand chemokine expression in inflammatory bowel disease, Inflamm Res.2001 50(10):491-5). Up-regulation of a membrane-bound cytokine receptorwas also observed in diseased bowel tissue from Crohn's patients(Holtmann M. H. et al., Tumor necrosis factor-receptor 2 is up-regulatedon lamina propria T cells in Crohn's disease and promotes experimentalcolitis in vivo, Eur J Immunol. 2002 32(11):3142-51). The collection ofthe tissue and mucosal samples requires the use of invasive andpotentially dangerous surgical techniques, thus limiting the practicalapplicability of these measurements for diagnostics. The diagnosticutility of these measurements is also unknown. In addition, at least onereport found that cytokine levels do not discriminate between Crohn'sdisease and ulcerative colitis (Banks C, et al., Chemokine expression inIBD. Mucosal chemokine expression is unselectively increased in bothulcerative colitis and Crohn's disease, J. Pathol. 2003 199(1):28-35).

Increased levels of IL-1β, IL-6 and soluble TNF receptor II wereobserved in stool samples from mice with chemically induced colitis(Lindsay J. O., et al., IL-10 gene therapy is therapeutic for dextransodium sulfate-induced murine colitis, Dig Dis Sci. 2004 49(7-8):1327-34). It is not clear whether similar effects occur in human CD andUC patients.

Up-regulation of cytokine levels in the bowel often does not lead tocorresponding changes in blood (Abstract of Kmiec Z., Cytokines ininflammatory bowel disease. Arch. Immunol. Ther. Exp. (Warsz). 199846(3): 143-55). In one study that did report a change in the serum levelof a cytokine in IBD, the average serum eotaxin levels for a populationof CD and UC patients was shown to be significantly different than theaverage value calculated for a normal population (Mir A, et al.,Elevated serum eotaxin levels in patients with inflammatory boweldisease, Am J. Gastroenterol. 2002 June; 97(6):1452-7). No statisticaldifference was observed between the CD and UC populations. The resultsshowed a significant overlap in the distribution of levels in the normaland diseased population; the serum level of eotaxin would therefore beexpected to be a relatively poor predictor of IBD.

SUMMARY OF THE INVENTION

Disclosed herein are inventive assay methods comprising measuring thelevels of one or more cytokines in the sample. In some embodiments ofthe invention, the method may involve determining from measured cytokinelevels if the patient has an inflammatory disease and/or determiningfrom measured cytokine levels, the level of inflammation due to aninflammatory disease and/or obtaining and measuring samples at differenttimes to monitor the progression of an inflammatory disease or theeffectiveness of treatments for such disease. In one embodiment, themethod includes measuring the level of sTNFRII. In certain embodiments,the method includes measuring a plurality of cytokines and may alsoinclude comparing the levels of these cytokines to cytokine profilesdetermined to be indicative of the disease. A variety of samples may beanalyzed. In certain embodiments, the samples may be obtained by anon-surgically invasive procedure from a human patient and may, forexample, include blood, serum, plasma, fecal, or urine samples.

In some embodiments of the invention, determining the presence or extentof disease may comprise comparing the levels of one or more cytokines tocytokine profiles indicative of the presence or extent of the disease.In one example, the levels of one or more cytokines in blood, serum andplasma samples are compared to cytokine profiles indicative of thepresence or extent of the disease. The step of comparing may comprisecomparing cytokine levels to detection cut-off values, comparing ratiosof cytokine levels to detection cut-off ratio values; comparing levelsof two cytokines to detection cut-off lines in correlation plots of thetwo analytes, comparing levels of multiple cytokines to detectioncut-off curves or surfaces in multi-analyte correlation plots and/orcomparing levels of multiple cytokines to detection zones (e.g.,detection areas or detection volumes) in multi-analyte correlationplots.

One embodiment of the invention includes a method for diagnosinginflammatory bowel disease comprising: measuring the level of sTNFRII ina sample, for example, a sample obtained from a patient suspected ofhaving inflammatory bowel disease; and diagnosing from the measuredlevel the presence or absence in the patient of inflammatory boweldisease. The cytokine (e.g. sTNFRII) level can be measured by a varietyof techniques available to a skilled artisan, including binding assaytechniques such as immunoassays, solid-phase binding assays and/oragglutination assays. Determination may comprise comparing this measuredlevel to a detection cut-off value, wherein the sTNFRII level above thedetection cut-off value is considered indicative of inflammatory boweldisease. In one embodiment, the sample is a blood, serum or plasmasample.

One embodiment of the invention involves a method for diagnosingulcerative colitis comprising: measuring the level of sTNFRII in asample, for example, a sample obtained from a patient suspected ofhaving ulcerative colitis; and diagnosing from the measured level thepresence or absence in said patient of ulcerative colitis. In oneembodiment, the sample is a blood, serum or plasma sample.

In another specific example, the cytokines for diagnosing ulcerativecolitis disease comprise one or more cytokines selected from the groupconsisting of RANTES, sIL-6R, sTNFRII, IL-1β, IL-13, and IL-6. Yet inanother specific example, the cytokines for diagnosing ulcerativecolitis disease are selected from one or more of the group consisting ofRANTES, sIL-6R, sTNFRII, and IL-1β.

In another specific example, the cytokines for diagnosing Crohn'sdisease comprise one or more cytokines selected from the list consistingof RANTES, sIL-6R, sTNFRII, IL-2, IL-4, IL-5, IL-8, and TNF. In yetanother specific example, the cytokines for diagnosing Crohn's diseasecomprise one or more cytokines selected from the group consisting ofRANTES, sIL-6R, and sTNFRII.

Certain embodiments of the methods of the invention may furtherdistinguish ulcerative colitis from Crohn's disease on the basis of themeasured level of a selected cytokine, e.g. the measured sTNFRII level.For example, distinguishing ulcerative colitis from Crohn's disease maycomprise comparing measured sTNFRII level to a discrimination cut-offvalue, wherein the measured level below the discrimination cut-off valueis considered indicative of Crohn's disease and above the discriminationcut-off value is considered indicative of ulcerative colitis.

In one embodiment of the present invention, the detection cut-off valueis set between 5 and 7 ng per ml of sample volume and the discriminationcut-off value is set between 8 and 10 ng per ml of sample volume.

One embodiment of the invention relates to a method for diagnosinginflammatory bowel disease comprising: measuring the level of a firstcytokine, for example, in a sample obtained from a patient suspected ofhaving inflammatory bowel disease; measuring the level of one or moreadditional cytokines, wherein the one or more additional cytokines aredifferent form the first cytokine; and diagnosing from the firstcytokine level and from the one or more additional cytokine levels thepresence of absence in said patient of inflammatory bowel disease. Inone embodiment, the sample is a sample collected via a non-surgicallyinvasive procedure from a patient. In certain cases, the samplecomprises a serum, plasma or blood sample. In other cases, the samplecomprises a fecal or urine sample.

According to one embodiment of the invention, diagnostically valuablecytokines may be selected from a group comprising IL-1β, IL-12p70,IL-10, IL-2, GM-CSF, TNF, IL-8, IL-4, IL-5, IL-6, Eotaxin, IFN-α, IFN-γ,sIL-6R, IL-12(total), IL-13, MIP-1β, MCP-1, RANTES and sTNFRII. In onespecific example, the cytokines are selected from the group consistingof IL-12p70, IL-10, IL-2, TNF, IL-8, IL-4, IL-5, IL-6, Eotaxin, sIL-6R,IL-12(total), MIP-1β, MCP-1, RANTES and sTNFRII. In another specificexample, the cytokines are selected from the group consisting ofEotaxin, sIL-6R, MIP-1β, MCP-1, and RANTES. In another specific example,the first cytokine is MCP-1 and the additional cytokine is MIP-1β

In one embodiment of the invention, sTNFRII is selected as the firstcytokine. Thus, the invention is a method for diagnosing inflammatorybowel disease comprising: measuring the sTNFRII level in a sample, forexample, a sample obtained from a patient suspected of havinginflammatory bowel disease; measuring one or more additional cytokines(e.g., levels of one or more of IL-1β, IL-12p70, IL-10, IL-2, GM-CSF,TNF, IL-8, IL-4, IL-5, IL-6, Eotaxin, IFN-α, IFN-γ, sIL-6R,IL-12(total), IL-13, MIP-1β, MCP-1 and/or RANTES); and diagnosing fromthe sTNFRII level and from one or more additional cytokine levels thepresence or absence of inflammatory bowel disease in the patient. In onespecific example, the additional cytokine(s) are selected from the groupconsisting of one or more of IL-12p70, IL-10, IL-2, TNF, IL-8, IL-4,IL-5, IL-6, Eotaxin, sIL-6R, IL-12(total), MIP-1β, MCP-1 and RANTES. Inanother specific example, the additional cytokines are selected from thegroup consisting of one or more of Eotaxin, sIL-6R, MIP-1β, MCP-1, andRANTES. In another specific example, the additional cytokine is MIP-1β.

Determining from the sTNFRII level and from one or more additionalcytokine levels if the patient has inflammatory bowel disease maycomprise comparing the sTNFRII level and one or more additional levelsto a cytokine profile determined to be indicative of inflammatory boweldisease. The step of comparing may comprise comparing cytokine levels todetection cut-off values, comparing ratios of levels to detectioncut-off ratio values and/or comparing levels to detection cut-off lines,curves or surfaces in multi-analyte correlation plots. In oneembodiment, a sNFRII level above a sTNFRII detection cut-off value and alevel of an additional cytokine below a cytokine detection cut-off valueare considered indicative of inflammatory bowel disease. In anotherembodiment, a ratio of the sTNFRII level to one additional cytokinelevel above a detection cut-off ratio value is considered indicative ofinflammatory bowel disease. In yet another embodiment, a sTNFRII levelabove a sTNFRII detection cut-off line is considered indicative ofinflammatory bowel disease. In yet another embodiment IL-6 is selectedas a first cytokine and IL-13 as a second cytokine.

One specific example of the invention also relates to a method fordiagnosing inflammatory bowel disease by measuring pair-wise cytokinelevel profiles selected from the group consisting of sTNFRII/RANTES,sTNFRII/sIL-6R, sIL-6R/RANTES, IL-5/sIL-6R and sTNFRII/IL-4.

According to one embodiment of the invention, the method may comprisedistinguishing ulcerative colitis from Crohn's disease for patientshaving an inflammatory bowel disease on the basis of a measured sTNFRIIlevel and one or more additional measured cytokine levels. In oneexample, the sTNFRII level and one or more additional cytokine levelsare blood, serum or plasma levels. For example, ulcerative colitis canbe distinguished from Crohn's disease, according to the invention, bycomparing the sTNFRII level and one or more additional cytokine levelsto profiles determined to be indicative of Crohn's disease or ulcerativecolitis. The step of comparing may comprise comparing levels todiscrimination cut-off values, comparing ratios of levels todiscrimination cut-off ratio values, and/or comparing levels todiscrimination cut-off lines. In one embodiment, ulcerative colitis isdistinguished from Crohn's disease by comparing the sTNFRII level to asTNFRII discrimination cut-off value, wherein a sTNFRII level below saidsTNFRII discrimination cut-off value is considered indicative of Crohn'sdisease and above the sTNFRII discrimination cut-off value is consideredindicative of ulcerative colitis. In yet another embodiment, ulcerativecolitis is distinguished from Crohn's disease by comparing the sTNFRIIlevel to a sTNFRII discrimination cut-off line, wherein sTNFRII levelbelow the sTNFRII discrimination cut-off line is considered indicativeof Crohn's disease and above the sTNFRII discrimination cut-off line isconsidered indicative of ulcerative colitis.

In yet another embodiment, ulcerative colitis is distinguished fromCrohn's disease by comparing a measured sTNFRII level to a cytokineprofile defined as areas situated between a first detection cut-off lineand a second discrimination cut-off line on a correlation plot.

In one embodiment, ulcerative colitis is distinguished from Crohn'sdisease by comparing two or more cytokines measured in a patient to aprofile of these two or more cytokines, e.g., values, ratios, lines orzones on the correlation plot, indicative of a patient having ulcerativecolitis, a patient having Crohn's disease and a healthy individual. Inone specific example, pare-wise cytokine profiles are selected from thegroup consisting of, but not limited to, sTNFRII/RANTES, sTNFRII/sIL-6R,and sTNFRII/IL-4.

Another embodiment of the invention relates to methods for measuring theextent of inflammation due to IBDs. The inventive methods may include anassay method comprising: measuring the level of sTNFRII in a sample, forexample, a sample obtained from a patient that has or is suspected tohave an inflammatory disease; and determining from the level of sTNFRIIthe extent of inflammation from the disease.

One embodiment of the invention includes a method comprising: measuringa level of a first cytokine, for example, measuring in a sample obtainedfrom a patient that has or is suspected to have an inflammatory disease,measuring the level of one or more additional cytokines, wherein the oneor more additional cytokines differ from the first cytokine; anddetermining from measured levels the extent of inflammation from thedisease. In one embodiment, the cytokines comprise one or more cytokinesselected from the group consisting of IL-1β, IL-12p70, IL-10, IL-2,GM-CSF, TNF, IL-8, IL-4, IL-5, IL-6, Eotaxin, IFN-α, IFN-γ, sIL-6R,IL-12(total), IL-13, MIP-1β, MCP-1, RANTES and sTNFRII. In anotherembodiment, the cytokines are selected from the group consisting ofIL-12p70, IL-10, IL-2, TNF, IL-8, IL-4, IL-5, IL-6, Eotaxin, sIL-6R,IL-12(total), MIP-1β, MCP-1, RANTES and sTNFRII. In another embodiment,the cytokines comprise one or more cytokines selected from the groupconsisting of Eotaxin, sIL-6R, MIP-1β, MCP-1, and RANTES. In anotherembodiment, the first cytokine is MCP-1 and the second cytokine isMIP-1β.

In yet another embodiment, the first cytokine is sTNFRII. In onespecific example of this embodiment, the additional cytokine(s) areselected from the group consisting of IL-12p70, IL-10, IL-2, TNF, IL-8,IL-4, IL-5, IL-6, Eotaxin, sIL-6R, IL-12 (total), MIP-1β, MCP-1 andRANTES. In another specific example, the additional cytokine(s) areselected from the group consisting of Eotaxin, sIL-6R, MIP-1.beta.,MCP-1, and RANTES. In another specific example, the additional cytokineis MIP-1β. In another specific example, a pair of cytokines is selectedfrom the group consisting of, but not limited to, sTNFRII/RANTES,sTNFRII/sIL-6R, and sTNFRII/IL-4.

Another embodiment of the invention relates to methods for monitoringthe progression or treatment of IBDs. The invention includes a methodfor monitoring the progression or treatment of an IBD comprising:measuring the levels of sTNFRII in samples obtained at different times,for example, samples obtained from a patient that has or is suspected tohave an inflammatory disease; and determining from the levels of sTNFRIIthe progression or efficacy of treatment of the disease.

One embodiment of the invention includes a method for monitoring theprogression or treatment of an IBD comprising: measuring the levels of afirst cytokine in samples obtained at different times, for example,samples obtained from a patient that has or is suspected to have aninflammatory disease; measuring the levels of one or more additionalcytokines in the samples from the same patient obtained at the sametimes as samples for the first cytokine, for example, the same samples,wherein the one or more additional cytokines differ from the firstcytokine; and determining from measured levels the progression orefficacy of treatment of the disease. In one embodiment, the cytokinescomprise one or more cytokines selected from the group consisting ofIL-1β, IL-12p70, IL-10, IL-2, GM-CSF, TNF, IL-8, IL-4, IL-5, IL-6,Eotaxin, IFN-α, IFN-γ, sIL-6R, IL-12(total), IL-13, MIP-1β, MCP-1,RANTES and sTNFRII. In another embodiment, the cytokines comprise one ormore cytokines selected from the group consisting of IL-12p70, IL-10,IL-2, TNF, IL-8, IL-4, IL-5, IL-6, Eotaxin, sIL-6R, IL-12 (total),MIP-1β, MCP-1, RANTES and sTNFRII. In another embodiment, the cytokinescomprise one or more cytokines selected from the group consisting ofEotaxin, sIL-6R, MIP-1β, MCP-1, and RANTES. In another embodiment, thefirst cytokine is MCP-1 and the second cytokine is MIP-1β.

In yet another embodiment, the first cytokine is sTNFRII. In onespecific example of this embodiment, the additional cytokine(s) areselected from the group consisting of IL-12p70, IL-10, IL-2, TNF, IL-8,IL-4, IL-5, IL-6, Eotaxin, sIL-6R, IL-12 (total), MIP-1β, MCP-1 andRANTES. In another specific example, the additional cytokine(s) areselected from the group consisting of Eotaxin, sIL-6R, MIP-1β, MCP-1,and RANTES. In another specific example, the additional cytokine isMIP-1β. In another specific example, a pair of cytokines is selectedfrom the group consisting of, but not limited to, sTNFRII/RANTES,sTNFRII/sIL-6R, and sTNFRII/IL-4.

Another aspect of the invention involves a method for evaluation of theeffectiveness of a drug or drug candidate for treating IBDs. Forexample, the invention includes a method for evaluating theeffectiveness of a drug and/or drug candidate comprising: exposing ahuman or non-human animal with IBD and/or a model system, for example, atissue, cell culture or a biochemical system, to the drug or drugcandidate; measuring the levels of sTNFRII in a sample obtained from thehuman or non-human animal or a model system; and determining from thelevel the effectiveness of the drug or drug candidate.

In another example, the invention includes a method for evaluating theeffectiveness of a drug or drug candidate comprising: exposing a humanor non-human animal with IBD and/or a model system, for example, atissue, cell culture or a biochemical system, to the drug or drugcandidate; measuring the level of a first cytokine in a sample obtainedfrom the human or non-human animal or a model system; measuring thelevel of one or more additional cytokines in the same sample or adifferent sample obtained from the same human or non-human animal or amodel system, wherein the one or more additional cytokines differ fromthe first cytokine; and determining from measured levels theeffectiveness of the drug or drug candidate. In one embodiment, thecytokines comprise one or more cytokines selected from the groupconsisting of IL-1, IL-12p70, IL-10, IL-2, GM-CSF, TNF, IL-8, IL-4,IL-5, IL-6, Eotaxin, IFN-α, IFN-γ, sIL-6R, IL-12(total), IL-13, MIP-1β,MCP-1, RANTES and sTNFRII. In another embodiment, the cytokines compriseone or more cytokines selected from the group consisting of IL-12p70,IL-10, IL-2, TNF, IL-8, IL-4, IL-5, IL-6, Eotaxin, sIL-6R, IL-12(total), MIP-1β, MCP-1, RANTES and sTNFRII. In another embodiment, thecytokines comprise one or more cytokines selected from the groupconsisting of Eotaxin, sIL-6R, MIP-1β, MCP-1, and RANTES. In anotherembodiment, the first cytokine is MCP-1 and the second cytokine isMIP-1β. In yet another embodiment, the first cytokine is sTNFRII. In onespecific example of this embodiment, the additional cytokine(s) areselected from the group consisting of IL-12p70, IL-10, IL-2, TNF, IL-8,IL-4, IL-5, IL-6, Eotaxin, sIL-6R, IL-12(total), MIP-1β, MCP-1 andRANTES. In another specific example, the additional cytokine(s) areselected from the group consisting of Eotaxin, sIL-6R, MIP-1β, MCP-1,and RANTES. In another specific example, the additional cytokine isMIP-1β. In another specific example, a pair of cytokines is selectedfrom the group consisting of, but not limited to, sTNFRII/RANTES,sTNFRII/sIL-6R, and sTNFRII/IL-4.

The method may also include comparing the levels to the levels in acontrol human or non-human animal that was not treated with the drug ordrug candidate. The human or non-human animal in these drug evaluationmethods may be replaced with an in vitro IBD model system, for example,tissue, cell culture or biochemical systems that model the behavior ofIBDs.

Suitable samples for detecting or monitoring IBD include blood, serum,plasma, fecal matter, biopsy tissue, intestinal mucosa and urine.Advantageously, certain embodiments of the invention allow measurementin samples obtained via non-surgically invasive procedures, such as, forexample, blood, serum, plasma, fecal matter, and urine samples. Formethods involving the measurement of multiple cytokines, each cytokinemay be measured separately. Alternatively, multiplexed measurementapproaches (including approaches known in the art such as array-based orflow cytometry-based approaches) may be used to concurrently measuremultiple cytokines in a single volume of sample. Such multiplexedmeasurements may be advantageously carried out in a single assay chambersuch as a single test tube, a single well of an assay plate and/or anassay chamber of an assay cartridge.

Certain methods of the invention may be supplemented by conducting adiagnostic test to determine if the patient has viral or bacterialinfection. Certain methods of the invention may further compriseadministering to the tested patient an effective amount of drug foreffective treatment of the diagnosed IBD.

The invention also includes reagents and kits for carrying out themethods of the invention. In one embodiment, a kit comprises antibodiesagainst the cytokines being measured in a method of the invention. Thekit may further comprise assay diluents, standards, controls and/ordetectable labels. The assay diluents, standards and/or controls may beoptimized for a particular sample matrix. For example, for measurementsin blood, serum or plasma samples, the diluents, standards and controlsmay include i) human blood, serum or plasma; ii) animal blood, serum orplasma or iii) artificial blood, serum or plasma substitutes.

A variety of cytokines can potentially be useful as diagnostic marker(s)for performing the inventive methods for the diagnosis and/or monitoringof inflammatory bowel disease and for screening drugs or drug candidatesfor efficacy in treating inflammatory bowel disease. Indeed, asdescribed in more detail below, certain embodiments of the inventionprovide methods for determining the efficacy of particular candidatecytokine(s) for acting as such diagnostic marker(s). Using the methodsof the present invention, one of ordinary skill in the art will be ableto determine without undue experimentation the ability of one or moreselected cytokines, including cytokines not specifically listed hereinand indeed not yet discovered, to be useful as markers whose measuredlevels/profiles may be employed in performing the various diagnostic andscreening methods of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are schematic are not intended to be drawn toscale. In the figures, each identical, or substantially similar,component that is illustrated in various figures is typicallyrepresented by a single numeral or notation. For purposes of clarity,not every component is labeled in every figure, nor is every componentof each embodiment of the invention shown where illustration is notnecessary to allow those of ordinary skill in the art to understand theinvention. In the drawings:

FIGS. 1A-ID depict examples of possible distributions of assay signalsfor positive and negative samples (FIGS. 1A, 1B, 1C) for an assay andthe ROC curves that are generated in each case (FIG. 1D);

FIGS. 2A-2D depict ROC curves for using sTNFRII for Crohn's diseasedetection in plasma samples (FIG. 2A), Crohn's disease detection inserum samples (FIG. 2B), ulcerative colitis detection in plasma samples(FIG. 2C), and ulcerative colitis detection in serum samples (FIG. 2D);

FIG. 3 depicts sTNFRII levels (ng/mL, y axis) in serum for healthyindividuals, individuals diagnosed with Crohn's disease and individualsdiagnosed with ulcerative colitis;

FIG. 4 depicts profiles of sTNFRII levels (pg/mL, y axis) and MIP-1βlevels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 5 depicts profiles of sTNFRII levels (pg/mL, y axis) and RANTESlevels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 6 depicts profiles of sTNFRII levels (pg/mL, y axis) and MCP-1levels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 7 depicts profiles of sTNFRII levels (pg/mL, y axis) and sIL-6Rlevels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 8 depicts profiles of sTNFRII levels (pg/mL, y axis) and IL-8levels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 9 depicts profiles of sTNFRII levels (pg/mL, y axis) and Eotaxinlevels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 10 depicts profiles of sTNFRII levels (pg/mL, y axis) and IL-2levels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 11 depicts profiles of MCP-1 levels (pg/ml, y axis) and MIP-1βlevels (pg/ml, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 12 depicts profiles of RANTES levels (pg/mL, y axis) and MCP-1levels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 13 depicts profiles of RANTES levels (pg/mL, y axis) and MIP-1βlevels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 14 depicts profiles of MIP-1β levels (pg/mL, y axis) and Eotaxinlevels (pg/mL, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis;

FIG. 15 depicts profiles of sTNFRII levels (pg/ml, y axis) and MIP-1βlevels (pg/ml, x axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis; and

FIG. 16 depicts profiles of sTNFRII levels (pg/ml, x axis) and IL-4levels (pg/ml, y axis) in serum for healthy individuals, individualsdiagnosed with Crohn's disease and individuals diagnosed with ulcerativecolitis.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are inventive methods for conducting diagnostic testsfor the detection of inflammatory diseases such as inflammatory boweldisease (IBD). The diagnostic tests may comprise measuring analytes inbiological samples, for example measuring disease markers, markers ofinflammation, and/or cytokines, where the levels of the analytes areindicative of the presence or severity of an inflammatory disease. Oneaspect of the invention is identifying diagnostically valuable markersof IBDs, for example diagnostically valuable markers of Crohn's disease(CD) and ulcerative colitis (UC). Another aspect of the inventionrelates to methods for detecting and/or distinguishing various IBDs,such as CD and UC. Another aspect of the invention further relates tomethods for monitoring the progression or treatment of inflammatorybowel disease in a patient by administering and/or repetitivelyadministering the diagnostic tests according to the methods of thepresent invention. In one example, the diagnostic methods may be used toevaluate the effectiveness of a drug or drug candidate for treatinginflammatory diseases by measuring the effect of the drug or drugcandidate on the levels of disease-specific analytes in samples frompatients, animal models, tissue samples and cell cultures treated with adrug or a drug candidate. Another aspect of the invention providesmethods for determining the efficacy of particular candidate analytes,such as particular cytokine(s), for acting as diagnostic marker(s) inthe inventive methods for the diagnosis and/or monitoring ofinflammatory bowel disease and for screening drugs or drug candidatesfor efficacy in treating inflammatory bowel disease.

Analytes that may be measured using the assay methods of the presentinvention include inflammatory markers, such as cytokines, secretedproteins that are involved in regulation of immune response. Cytokinesinclude the interleukins (ILs), interferons (IFNs), chemokines, tumornecrosis factors (TNFs), and a variety of colony stimulating factors(CSFs). The term cytokines, as used herein, also includes solublecytokine receptors. Specific cytokines that may be measured in theassays of the invention include, but are not limited to, cytokineslinked to TH1 response, cytokines linked to TH2 response,pro-inflammatory cytokines and/or cytokines selected from the groupconsisting of IL-1β, IL-12p70, IL-10, IL-2, granulocyte-macrophagecolony stimulating factor (GM-CSF), TNF-α, IL-8, IL-4, IL-5, IL-6,Eotaxin, IFN-α, IFN-γ, soluble IL-6 receptor (sIL-6R), IL-12(total),IL-13, MIP-1β, MCP-1, RANTES and soluble TNF-β receptor II (sTNFRII).According to one aspect of the invention, analytes could advantageouslymeasured in a sample obtained via a non-surgically invasive collectiontechnique, such as in a blood, serum, plasma, fecal, or urine samplefrom a patient having, or suspected to have an IBD.

According to one aspect of the invention, the levels of cytokine orother disease marker candidates are measured in the samples collectedfrom individuals clinically diagnosed with Crohn's disease andulcerative colitis (e.g., using conventional diagnostic methods, such asdoctor's interview, endoscopies, imaging and/or biopsy) and from healthyindividuals. Within non-limiting examples of this invention, specificcytokines valuable as a marker for distinguishing between normal anddiseased patients could be identified using visual inspection of thedata, for example, data plotted on a one-dimensional ormulti-dimensional graph, or by using methods of statistical analysis,such as a statistically weighted difference between control individualsand diseased patients and/or Receiver Operating Characteristic (ROC)curve analysis.

For example in one exemplary embodiment of the present invention,diagnostically valuable cytokines may be first identified using astatistically weighted difference between control individuals anddiseased patients, calculated as

$\frac{D - N}{\sqrt{\sigma_{D}*\sigma_{N}}}$

where D is the median concentration of a cytokine in patients diagnosedas having, for example, ulcerative colitis or Crohn's disease, N is themedian of the control individuals, σ_(D) in the standard deviation of Dand σ_(N) is the standard deviation of N. The larger the magnitude, thegreater the statistical difference between the diseased and normalpopulations.

According to one embodiment of the invention, cytokines resulting in astatistically weighted difference between control individuals anddiseased patients of greater than 0.2, 0.5, 1, 1.5, 2, 2.5 or 3 could beidentified as diagnostically valuable markers.

Another method of statistical analysis that can be useful in theinventive methods of the invention for determining the efficacy ofparticular candidate analytes, such as particular cytokine(s), foracting as diagnostic marker(s) is Receiver Operating Characteristic(ROC) curve analysis. An ROC curve is a graphical approach to looking atthe effect of a cut-off criterion (e.g., a cut-off value for adiagnostic indicator such as an assay signal or the level of an analyte)on the ability of a diagnostic to correctly identify positive andnegative samples or subjects. (See, FIGS. 1A-1D) One axis of the ROCcurve is the true positive rate (TPR, the probability that a truepositive sample/subject will be correctly identified as positive) or,alternatively, the false negative rate (FNR=1−TPR, the probability thata true positive sample/subject will be incorrectly identified as anegative). The other axis is the true negative rate (TNR, theprobability that a true negative sample will be correctly identified asa negative) or, alternatively, the false positive rate (FPR=1−TNR, theprobability that a true negative sample will be incorrectly identifiedas positive). The ROC curve is generated using assay results for apopulation of samples/subjects by varying the diagnostic cut-off valueused to identify samples/subjects as positive or negative and plottingcalculated values of TPR (or FNR) and TNR (or FPR) for each cut-offvalue. The area under the curve (referred to herein as the ROC area) isone indication of the ability of the diagnostic to separate positive andnegative samples/subjects.

FIGS. 1A-1C show examples of three different distributions of assaysignals for positive and negative samples, and FIG. 1D shows the ROCcurves that are generated in each case by varying the cut-off valueacross the range of possible assay signals. A diagnostic assay thatalmost perfectly separates the negative and positive populations FIG.1A) will give an ROC area approaching 1.0. A diagnostic that providesalmost no separation of negative and positive populations (FIG. 1C) willgive an ROC area approaching 0.5. A diagnostic assay that provides anintermediate level of separation (FIG. 1B) will give an ROC areasomewhere in the middle between 0.5 and 1.0. For example, FIGS. 2A-2Ddepict sTNFRII ROC curves for using TNFRII levels as a diagnostic forCrohn's disease detection using plasma samples (FIG. 2A), Crohn'sdisease detection using serum samples (FIG. 2B), ulcerative colitisdetection using plasma samples (FIG. 2C), and ulcerative colitisdetection using serum samples (FIG. 2D).

ROC curve analysis can also provide an approach to selecting a cut-offvalue that best optimizes the trade-off between TPR and FPR to meet theobjectives of a specific application. One approach is to select thecut-off value that maximizes the product of the TPR and TNR, althoughfor certain applications the TPR or TNR may be held more important andweighted more heavily. A more detailed explanation of how ROC analysiscan be used to select cut-off values yielding a desired level ofstatistical confidence for correct diagnosis is presented in M H Zweig,G Campbell, Receiver-operating characteristic (ROC) plots: a fundamentalevaluation tool in clinical medicine, Clin Chem 1993: 39(4); 561-77; P.A. Murtaugh, ROC curves with multiple marker measurements, Biometrics1995: 51; 1514-22, each of which is incorporated, herein, by thisreference.

Diagnostic indicators analyzed by ROC curve analysis may simply be alevel of an analyte (e.g., a cytokine) or an assay signal.Alternatively, the diagnostic indicator may be a function of multiplemeasured values, for example, a function of the level/assay signal of aplurality of analytes (e.g., a plurality of cytokines) or a functionthat combines the level/assay signal of one or more analytes with apatient scoring value that is determined based on visual, radiologicaland/or histological evaluation of a patient. The multi-parameteranalysis may provide more accurate diagnosis relative to analysis of asingle marker.

Candidates for a multi-analyte panel could be selected by using criteriasuch as individual analyte ROC areas, median difference between groupsnormalized by geometric interquartile range (IQR) etc. The objective isto partition the analyte space so as to improve separation betweengroups (for e.g. normal and disease populations) or to minimize themisclassification rate.

One approach is to define a panel response as a weighted combination ofindividual analytes and then compute an objective function like ROCarea, product of sensitivity and specificity etc. See, for e.g., PCTApplication No. WO 2004/058055, “Method and System for Disease DetectionUsing Marker Combinations”. The weighting coefficients define thepartitioning object; for linear combinations the object is a line in 2dimensions, a plane in 3 dimensions and a hyperplane in higherdimensions. The optimal coefficients maximize the objective function andcan be determined using algorithms for finding function extrema inmultiple dimensions—gradient descent methods, downhill simplex methods,simulated annealing and the like; more details can be found in“Numerical Recipes in C, The Art of Scientific Computing”, W. Press etal., Cambridge University Press, 1992.

Another approach is to use discriminant analysis, where a multivariateprobability distribution (normal, multinomial etc.) is used to describeeach group. Several distributions result in partitioning hyperplanes inanalyte space. One advantage of this approach is the ability to classifymeasurements into multiple groups (e.g. normal, disease 1, disease 2)simultaneously, rather than two at a time. For further details, see“Principles of Multivariate Analysis, A User's Perspective”, W. J.Krzanowski, Oxford University Press, 2000 and “MultivariateObservations”, G. A. F. Seber, John Wiley, 2004. Once the partitioninghyperplanes have been determined, the robustness of different assaypanels can be compared by evaluating a distance metric to the separatinghyperplanes for each group.

A skilled artisan will readily recognize that because the algorithmsdescribed above could be used to find the best classification betweengroups; they could also be used for distinguishing between differentdiseases or subgroups of the same disease. Finally, categorical data(age, gender, race etc) can also be coded into different levels and usedas an optimizing variable in this process.

One indication (the ROC area) of the diagnostic utility of a selectedgroup of exemplary markers is presented in Table 1.

TABLE 1 Disease (sample) Assay ROC Area Crohns (plasma) RANTES 0.86Crohns (serum) RANTES 1 Crohns (plasma) IL-6R 0.94 Crohns (serum) IL-6R0.87 Crohns (plasma) TNF-RII 0.93 Crohns (serum) TNF-RII 0.88 Crohns(plasma) IL-2 0.69 Crohns (serum) IL-2 0.76 Crohns (plasma) IL-4 0.7Crohns (serum) IL-4 0.75 Crohns (plasma) IL-5 0.77 Crohns (serum) IL-50.68 Crohns (plasma) IL-8 0.77 Crohns (serum) IL-8 0.64 Crohns (plasma)TNF 0.58 Crohns (serum) TNF 0.77 Crohns (plasma) MIP-1β 0.58 Crohns(serum) MIP-1β 0.98 Crohns (plasma) MCP-1 0.68 Crohns (serum) MCP-1 0.89Crohns (plasma) Eotaxin 0.56 Crohns (serum) Eotaxin 0.78 Crohns (plasma)IL-1β 0.91 Crohns (serum) IL-1β 0.55 Crohns (plasma) IFN-γ 0.79 Crohns(serum) IFN-γ 0.55 UC (plasma) TNF-RII 1 UC (serum) TNF-RII 1 UC(plasma) IL-6R 0.93 UC (serum) IL-6R 0.87 UC (plasma) IL-1β 0.89 UC(serum) IL-1β 0.85 UC (plasma) RANTES 0.88 UC (serum) RANTES 0.97 UC(plasma) IL-13 0.79 UC (serum) IL-13 0.97 UC (plasma) IL-6 0.75 UC(serum) IL-6 0.81 UC (plasma) MIP-1β 0.56 UC (serum) MIP-1β 0.98 UC(plasma) MCP-1 0.74 UC (serum) MCP-1 0.92 UC (plasma) Eotaxin 0.52 UC(serum) Eotaxin 0.83 UC (plasma) IFN-γ 0.82 UC (serum) IFN-γ 0.58

According to one embodiment of the invention, cytokines are selected sothat the ROC areas exceed, for example, 0.6, 0.7, 0.8 or 0.9. Thus, inone non-limiting example, ulcerative colitis is diagnosed using one ormore cytokines selected from the group consisting of RANTES, sIL-6R,sTNFRII, IL-1β, IL-13, IL-6 where the ROC area exceeds 0.6. In yetanother specific example, ulcerative colitis is diagnosed using one ormore cytokines selected from the group consisting of RANTES, sIL-6R,sTNFRII, IL-1β where the ROC area exceeds 0.8. In another specificexample, Crohn's disease is diagnosed using one or more of the cytokinesselected from the group consisting of RANTES, sIL-6R, sTNFRII, IL-2,IL-4, IL-5, IL-8, TNF, where the ROC area exceeds 0.6. In yet anotherspecific example, Crohn's disease is diagnosed using one or morecytokines selected from the group consisting of RANTES, sIL-6R, sTNFRII,where the ROC area exceeds 0.8.

According to one aspect of the invention, a pair-wise profile of twocytokines is used as a marker for IBDs. According to another embodimentof the invention, the cytokines are selected so that the multi-analyteROC area exceeds, for example, 0.6, 0.7, 0.8 or 0.9. By the way ofnon-limiting examples, ulcerative colitis and/or Crohn's disease isdiagnosed using a pair of cytokines selected from the list consisting ofTNFRII/RANTES, TNFRII/IL-6R, IL-6R/RANTES, IL-5/IL-6R or TNFRII/IL-4,where the ROC area exceeds 0.8. In certain embodiments, bothstatistically weighted difference analysis and ROC analysis are used toidentify diagnostically valuable cytokines of this invention.

In certain embodiments of the invention, sTNFRII is determined to beuseful as a marker for IBDs for certain methods of the invention. In oneembodiment, sTNFRII is used as a marker for Crohn's disease (CD) andulcerative colitis (UC). FIG. 3 shows that normal and IBD (UC or CD)populations can be distinguished based on the circulating levels ofsTNFRII as measured in blood/serum or plasma samples. As determinedaccording to the invention, measurement of sTNFRII levels may be used todiagnose IBD, monitor the progression of disease or the effectiveness oftherapy in a patient having IBD, and/or assess the effectiveness ofdrugs or drug candidates targeting IBDs. Consequently, one embodiment ofthe invention is a method for diagnosing inflammatory bowel diseasecomprising: measuring the level of sTNFRII in a sample, for example, asample obtained from a patient suspected of having inflammatory boweldisease; and diagnosing from the level of sTNFRII the presence orabsence in the patient of inflammatory bowel disease. Furthermore, themethod may comprise an additional step of administering to the testedpatient an effective amount of drug for effective treatment for an IBD.

Another embodiment of the invention involves an assay method comprising:measuring the level of sTNFRII in a sample, for example, a sampleobtained from a patient suspected of having inflammatory bowel disease;and determining from the level of sTNFRII the extent of inflammation dueto the IBD.

Yet another embodiment is a method for specifically diagnosingulcerative colitis comprising: measuring the level of sTNFRII in asample, for example, a sample obtained from a patient suspected ofhaving UC; and diagnosing from the level of sTNFRII the presence orabsence in the patient of ulcerative colitis.

Yet another embodiment is a method for monitoring the progression ortreatment of IBDs comprising: measuring the levels of sTNFRII in thesamples, for example, the samples obtained at different times from apatient that has or is suspected to have an IBD; and determining fromthe level of sTNFRII the progression or efficacy of treatment of thedisease.

Yet another embodiment is a method for evaluation of the effectivenessof a drug or drug candidate for treating IBDs comprising: exposing ahuman or non-human animal with IBD, or a model system, such as a tissue,a cell culture or a biochemical system, to the drug or drug candidate;measuring the levels of sTNFRII in a sample, for example, a sampleobtained from the human or non-human animal or a model system; anddetermining from the level the effectiveness of the drug or drugcandidate. The human or non-human animal in these drug evaluationmethods may be replaced with an in vitro IBD model system, for example,tissue, cell culture or biochemical systems that model the behavior ofIBDs.

According to one embodiment, the determination if the patient hasinflammatory bowel disease from the level of sTNFRII may comprisecomparing the level of sTNFRII to a detection cut-off value (see, e.g.,cut-off value 300 in FIG. 3). In the example shown in FIG. 3, a sTNFRIIlevel above the detection cut-off value is considered indicative ofinflammatory bowel disease. The detection cut-off value is determinedfrom evaluation of the TNFRII levels in patients diagnosed with IBD ascompared to healthy individuals. The cut-off value 300 is determined byvisually evaluating test data in FIG. 3. A skilled artisan can readilydetermine an appropriate cut-off value from the data, either visually,or according to other available techniques, for example as described inBoyd J. C. “Reference Limits in the Clinical Laboratory” in ProfessionalPractice in Clinical Chemistry: A Companion Text; D. R. Dufour Ed.,1999, Washington D.C.: American Assoc. Clin. Chem., Chapter 2, pp. 2-1to 2-7, incorporated, herein, by this reference. For background on theselection of decision limits (i.e., cut-offs) or the calculation, fromtest results, of disease likelihood see Boyd J. C. “Statistical Aids forTest Interpretation” in Professional Practice in Clinical Chemistry: ACompanion Text; D. R. Dufour Ed., 1999, Washington D.C.: American Assoc.Clin. Chem., Chapter 3, pp. 3-1 to 3-11, U.S. patent application Ser.No. 10/410,572, “System and Method for Identifying a Panel ofIndicators,” filed on Apr. 8, 2002, published as U.S. Pat. Publ. No.20040121350, U.S. patent application Ser. No. 10/331,127, “Method andSystem for Disease Detection Using Marker Combinations,” filed on Dec.27, 2002, published as U.S. Pat. Publ. No. 20040126767, U.S. patentapplication Ser. No. 10/603,891, “Markers for Differential Diagnosis AndMethods of Use Thereof,” filed on Jun. 23, 2003, published as U.S. Pat.Publ. No. 20040253637, all of which are incorporated, herein, by thisreference.

According to another aspect of the invention, a system for identifying apanel of markers for diagnosis of a disease or a condition includesmeans for calculating a panel response for each patient in a set ofdiseased patients and in a set of non-diseased patients. In oneembodiment the panel response is a function of a value of each of aplurality of markers in a panel of markers. The means for calculatingmay be a central processing unit (CPU), as may be available on a desktopcomputer, a laptop computer, a workstation or a mainframe, for example.

In a preferred embodiment, one or more ‘derived markers’, which are afunction of one or more measured markers, may be incorporated into theset of markers being studied. For example, derived markers may berelated to the change in one or more measured marker values, or may berelated to a ratio of two measured marker values. In many diseases therewill be rapid change in marker value some time after an event. Forexample, following an acute myocardial infarction, (AMI), myoglobin mayrise rapidly and peak about 3 hours from the event. It may then decayback to its nominal value. Looking for changes in markers can bepowerful diagnostic tool. Thus, the change in myoglobin over a period ofan hour, for example, may be used as a “marker” in the panel.

The sTNFRII levels, in the context of FIG. 3, refers to theconcentration of the analyte as measured in a specific assay setting (inthis case, measurement on a SECTOR™ Imager 6000 reader (Meso ScaleDiscovery, a division of Meso Scale Diagnostics, LLC, Gaithersburg, Md.)using kits for multiplexed measurements of cytokines (Meso ScaleDiagnostics, LLC, Gaithersburg, Md.). The exact values of the measuredlevels and associated cut-off values may vary somewhat depending theexact assay conditions and the standards used for assay calibration andnatural variation between population groups, however, the general trends(e.g., relationship between the level of the sTNFRII and the diseasestate) should hold for different assays kits and assay instruments. Oneof ordinary skill in the art will be able to determine cut-off valuesapplicable for a specific set of assay conditions.

Within the confines of the experimental conditions used to generate thedata in FIG. 3 and set forth in the Examples below, the detectioncut-off value for sTNFRII (300) is between 5 and 7 ng/ml-determined byvisually examining data in FIG. 3. Given the teachings of the presentinvention and the knowledge of those of ordinary skill in the art, askilled artisan will readily recognize that the detection cut-off valuealso determines the selection stringency. The higher the cut-off valueselected, the lower is the chance that a false positive diagnosis, i.e.,diagnosing a healthy individual with an IBD, but the higher is a chanceof a false negative diagnosis, i.e., the chance of not detecting an IBD,and vice versa. Thus, in one embodiment of the invention a plurality ofdetection cut-off values could be determined to yield varying degrees ofdiagnostic confidence, e.g., definite diseased state, probable diseasedstate, low probability disease state, etc.

Given the teachings of the present invention and the knowledge of thoseof ordinary skill in the art, the cut-off value could be selected by anindividual performing the inventive methods, equipment manufacturer or astandard setting organization, for example, FDA by visually analyzingdata or with the assistance of the methods of statistical analysisreadily available to a skilled artisan, as described above.

In one embodiment, the invention also relates to methods thatdistinguish ulcerative colitis from Crohn's disease by comparing acytokine level, e.g. a sTNFRII level to a cytokine cut-off value, e.g. asTNFRII discrimination cut-off value (see, e.g., cut-off value 301 inFIG. 3), determined according to the invention to distinguish ulcerativecolitis from Crohn's disease. In the example shown in FIG. 3, a sTNFRIIlevel above the sTNFRII discrimination cut-off value (301) is consideredindicative of ulcerative colitis. A sTNFRII level below the sTNFRIIdiscrimination cut-off value (301) but above the pre-determineddetection cut-off value (300) is considered indicative of Crohn'sdisease.

Within the confines of the instrumentation and experimentationconditions used to generate the data in FIG. 3, the discriminationcut-off value for sTNFRII is between 8 and 10 ng/ml, as determined byvisual examination of data in FIG. 3. Selection of a plurality ofdiscrimination cut-off values allows adjustment in stringency ofdiscrimination. The higher the discrimination cut-off value selected,the lower is the chance that a false positive diagnosis, i.e.,diagnosing UC in a healthy individual or an individual having CD, butthe higher is a chance of a false negative diagnosis, i.e., the chanceof not diagnosing UC, and vice versa. When the detection cut-off valueis set at the discrimination cut-off value, the methods of the inventiondetect only patients having ulcerative colitis.

According to certain embodiments, the methods of the present inventionare also well suited for measuring plurality of analytes that may bepresent in a sample, thus, under some circumstances providing improvedconfidence of detection and discrimination of the diseases. Methods ofthe present invention are also well suited for measuring plurality ofanalytes in blood, serum or plasma samples of a patient.

In one embodiment, the invention relates to a method for diagnosinginflammatory bowel disease comprising: measuring the level of a firstcytokine, wherein, for example, the sample is obtained from a patientsuspected of having inflammatory bowel disease; measuring the level ofone or more additional cytokines in the same sample or a differentsample from the patient, wherein the one or more additional cytokinesare different form the first cytokine; and diagnosing from the firstcytokine level and from the one or more additional cytokine levels thepresence or absence in said patient of inflammatory bowel disease. Inone embodiment, the sample is a serum, plasma or blood sample. Inanother embodiment, the sample is a fecal or urine sample. Cytokinesthat may be measured in the methods of the invention include, but arenot limited to, cytokines linked to TH1 response, cytokines linked toTH2 response, pro-inflammatory cytokines and/or cytokines selected fromthe group consisting of IL-13, IL-12p70, IL-10, IL-2, GM-CSF, TNF, IL-8,IL-4, IL-5, IL-6, Eotaxin, IFN-α, IFN-γ, sIL-6R, IL-12(total), IL-13,MIP-1β, MCP-1, RANTES and sTNFRII. In one embodiment, the cytokines areselected from the group consisting of IL-12p70, IL-10, IL-2, TNF, IL-8,IL-4, IL-5, IL-6, Eotaxin, sIL-6R, IL-12(total), MIP-1β, MCP-1, RANTESand sTNFRII. In another embodiment, the cytokines are selected from thegroup consisting of Eotaxin, sIL-6R, MIP-1β, MCP-1, and RANTES. In oneembodiment, the first cytokine is MCP-1 and the additional cytokine isMIP-1B. In another embodiment, the first cytokine is sTNFRII. In onespecific example of this embodiment, the additional cytokine(s) areselected from the group consisting of IL-12p70, IL-10, IL-2, TNF, IL-8,IL-4, IL-5, IL-6, Eotaxin, sIL-6R, IL-12(total), MIP-1β, MCP-1 andRANTES. In another specific example, the additional cytokine(s) areselected from the group consisting of Eotaxin, sIL-6R, MIP-1β, MCP-1,and RANTES. In another specific example, the additional cytokine(s) areselected from the group consisting of sIL-6R and RANTES. In anotherspecific example, the additional cytokine is MIP-1β. In anotherembodiment, the first cytokine is sTNFRII and the additional cytokine isIL-4.

The cytokine measurements may also be used to determine the extent ofinflammation due to IBD by comparing the levels of one or more cytokinesin the patient diagnosed with an IBD to a cytokine level profile forhealthy individuals, or by comparing the levels of one or more cytokinesin the patient diagnosed with an IBD, where the samples are collected atdifferent times. Thus, the assay methods are particularly advantageousfor patient monitoring, treatment monitoring, or evaluation of theeffectiveness of a drug or drug candidate.

In one embodiment of the invention, sTNFRII is selected as the firstcytokine. The determining step may comprise comparing the measuredlevels of sTNFRII and one or more additional cytokines to a cytokineprofile determined to be indicative of inflammatory bowel disease. Theprofile can be created for a combination of analytes using measurementsof cytokine levels in diseased individuals as compared to healthyindividuals. The profile may be a pair-wise correlation profile or amultivariable correlation profile. Examples of the pair-wise correlationprofiles include, but not limited to, profiles exemplified by FIGS.4-16.

In some cases, ratios of the levels of two analytes can be used todiagnose disease and/or discriminate between diseases. FIG. 4 shows thecorrelation of serum sTNRFII and MIP-1β for normal and diseasedpopulations and shows that a ratio of the sTNFRII level to the MIP-1βlevel above a detection cut-off ratio value (i.e., the slope of line 400through the origin) is indicative of the inflammatory bowel disease. Thecut-off ratio value was selected by visual examination of data in FIG.4, however, a skilled artisan can readily understand that the cut-offratio value could also be selected using methods of statistical analysisknown in the art. Alternatively, a curve (e.g., a line) in a plotshowing the correlation of two analytes can be used for diagnosis and/ordiscrimination. FIG. 6 shows the correlation of serum levels sTNRFII andMCP-1 and shows that a sample that gives a point in the correlation plotwith a sTNFRII level above a sTNFRII detection cut-off line (600, inthis specific example, drawn by visual examination of data) isindicative in inflammatory bowel disease. Similarly, cut-off surfacescan be defined in 3 or more dimensional multi-analyte correlations.Combinations of cut-off values, lines or other curves (or surfaces)and/or ratios can be used to improve diagnosis. FIG. 5 shows acorrelation of sTNFRII and RANTES levels and shows that the combinationof a sTNFRII level above a TNFRII detection cut-off value (500) andRANTES level below a detection cut-off value (501) is indicative ofinflammatory bowel disease. In certain embodiments, values for multiplecytokine levels are analyzed to determine if they fall withinpre-determined detection zones in a correlation plot, the detectionzones being areas (in 2-D plots) or volumes (in higher dimensionalplots) that are indicative of a disease state. FIG. 9 shows acorrelation of sTNFRII and Eotaxin levels and shows that levels of thetwo cytokines that fall within a detection zone 900 are indicative ofinflammatory bowel disease.

According to one embodiment of the invention, the assay methods mayfurther distinguish ulcerative colitis from Crohn's disease on the basisof a measured sTNFRII level and one or more additional measured cytokinelevels. Ulcerative colitis may be distinguished from Crohn's disease bycomparing measured levels to profiles indicative of Crohn's disease orulcerative colitis. By way of example, FIG. 4 is a correlation plot thatshows a clear separation of normal, UC and CD populations.

Discrimination can be achieved through discrimination cut-off values,lines, ratios and/or zones indicative of Crohn's disease or ulcerativecolitis. FIG. 7 shows the correlation of sTNFRII with sIL-6R and showsthat sTNFRII levels that lie above discrimination cut-off line 700 areindicative of UC, while sTNFRII levels that lie between discriminationcut-off line 700 and detection cut-off line 701 are indicative of CD,and sTNFRII levels that fall below detection cut-off line 701 areindicative of normal subjects. FIG. 15 shows a correlation plot forsTNFRII and MIP-1β and shows that the UC and CD populations fallpredominantly into areas 1502 and 1503, respectively, defined by a firstdetection cut-off line 1500 and a second discrimination cut-off line1501 on the correlation plot.

Comparison of measured cytokine values to cytokine profiles may be usedto not only provide yes-no answers but also to predict the probabilityof disease or the confidence in a measurement, for example as discussedabove for ROC area analysis. For example, multiple cut-off values,ratios, lines, zones, etc. can be defined that provide different levelsof statistical confidence to a diagnostic conclusion. By way of example,FIGS. 8 and 10 have disease specific zones 800, 801, 1000 and 1001 thatare shown as contours, the different contour levels providing differentdegrees of sensitivity and specificity. The contour level may beselected to correspond to the probability of the disease and/orconfidence level of diagnostics. The contours in FIGS. 8 and 10 weredefined visually, but a skilled artisan will readily recognize that thecounters on a contour plot could be defined by using one of the abovediscussed methods of statistical analysis.

Certain embodiments of the invention that are applicable to methods fordetermining if the patient has inflammatory bowel disease anddistinguishing various IBDs may also comprise comparing the levels of afirst cytokine selected from the group consisting of sTNFRII, IL-1β,IL-12p70, IL-10, IL-2, GM-CSF, TNF, IL-8, IL-4, IL-5, IL-6, Eotaxin,IFN-α, IFN-γ, sIL-6R, IL-12(total), IL-13, MIP-1β, MCP-1 and RANTES andone or more additional cytokines selected from the same group to acytokine profile indicative of the inflammatory bowel disease. In oneembodiment, the cytokines are selected from the group consisting ofsTNFRII, Eotaxin, sIL-6R, MIP-1β, MCP-1, and RANTES. In anotherembodiment, the first cytokine is MCP-1 and the additional cytokine isMIP-1β. In another embodiment, the first cytokine is sTNFRII and theadditional cytokine is IL-4. The profile can be created for a specificcombination of analytes by measuring cytokine levels in diseasedindividuals and in healthy individuals. Examples of the profilesinclude, but not limited to, profiles exemplified by FIGS. 11-16. FIG.11 shows one example where individual cytokines measurements do notcompletely distinguish between normal and diseased populations (becauseof overlap in the distributions), but use or two cytokines provides acut-off line that provides excellent to separation of the populations.

One of ordinary skill in the art of diagnostic assays and statisticalanalysis of data, given the teaching and guidance provided herein, willbe able to select without undue burden appropriate cut-off values,lines, ratios, zones etc. for best meeting the needs (e.g., sensitivityand specificity) for a particular application. A variety of statisticaltools, such as, for example, receiver operating characteristic (ROC)curves, are available for evaluating the effect of adjustments tocut-offs on assay performance (e.g., predicted true positive fraction,false positive fraction, true negative fraction and false negativefraction). Alternatively, statistical analysis of patient populationscan allow conversion of specific analyte values into probabilities thatthe patient has or does not have a disease. For background on theselection and analysis of populations of individuals so as to determinereference ranges see Boyd J. C. “Reference Limits in the ClinicalLaboratory” in Professional Practice in Clinical Chemistry: A CompanionText; D. R. Dufour Ed., 1999, Washington D.C.: American Assoc. Clin.Chem., Chapter 2, pp. 2-1 to 2-7. For background on the selection ofdecision limits (i.e., cut-offs) or the calculation, from test results,of disease likelihood see Boyd J. C. “Statistical Aids for TestInterpretation” in Professional Practice in Clinical Chemistry: ACompanion Text; D. R. Dufour Ed., 1999, Washington D.C.: American Assoc.Clin. Chem., Chapter 3, pp. 3-1 to 3-11.

Given the teachings of the present invention, a skilled artisan willalso recognize that the choice of first cytokine and one or moreadditional cytokines may transpose correlation plot axes andconsequently the criteria for determining whether measured cytokinelevels of a patient's samples falling above or below particular cut-offratios, lines and/or profiles is indicative of a disease state and willbe able to adjust the analysis accordingly.

The cytokine levels may be measured using any of a number of techniquesavailable to the person of ordinary skill in the art, e.g., directphysical measurements (e.g., mass spectrometry) or binding assays (e.g.,immunoassays, agglutination assays, and immunochromatographic assays).The method may also comprise measuring a signal that results from achemical reaction, e.g., a change in optical absorbance, a change influorescence, the generation of chemiluminescence orelectrochemiluminescence, a change in reflectivity, refractive index orlight scattering, the accumulation or release of detectable labels fromthe surface, the oxidation or reduction or redox species, an electricalcurrent or potential, changes in magnetic fields, etc. Suitabledetection techniques may detect binding events by measuring theparticipation of labeled binding reagents through the measurement of thelabels via their photoluminescence (e.g., via measurement offluorescence, time-resolved fluorescence, evanescent wave fluorescence,up-converting phosphors, multi-photon fluorescence, etc.),chemiluminescence, electrochemiluminescence, light scattering, opticalabsorbance, radioactivity, magnetic fields, enzymatic activity (e.g., bymeasuring enzyme activity through enzymatic reactions that cause changesin optical absorbance or fluorescence or cause the emission ofchemiluminescence). Alternatively, detection techniques may be used thatdo not require the use of labels, e.g., techniques based on measuringmass (e.g., surface acoustic wave measurements), refractive index (e.g.,surface plasmon resonance measurements), or the inherent luminescence ofan analyte.

Binding assays for measuring cytokine levels may use solid phase orhomogenous formats. Suitable assay methods include sandwich orcompetitive binding assays. Examples of sandwich immunoassays aredescribed in U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat. No.4,366,241 to Tom et al., both of which are incorporated herein byreference. Examples of competitive immunoassays include those disclosedin U.S. Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 toLiotta, and U.S. Pat. No. 5,208,535 to Buechler et al., all of which areincorporated herein by reference.

Multiple cytokines may be measured using a multiplexed assay format,e.g., multiplexing through the use of binding reagent arrays,multiplexing using spectral discrimination of labels, multiplexing byflow cytometric analysis of binding assays carried out on particles(e.g., using the Luminex system). Suitable multiplexing methods includearray based binding assays using patterned arrays of immobilizedantibodies directed against the cytokines of interest. Variousapproaches for conducting multiplexed assays have been described. Forexample, multiplexed testing is described in U.S. patent applicationSer. Nos. 10/185,274 and 10/185,363, both filed on Jun. 28, 2002,entitled “Assay Plates, Reader Systems and Methods For Luminescence TestMeasurements,” published as U.S. Pat. Publ. No. 20040022677 andUS20050052646, respectively, U.S. patent application Ser. No.10/238,960, filed Sep. 10, 2002, entitled “Methods, Reagents, Kits andApparatus for Protein Function,” published as U.S. Pat. Publ. No.20030207290, U.S. patent application Ser. No. 10/238,391, filed Sep. 10,2002, entitled “Methods and apparatus for conducting multiplemeasurements on a sample”; published as U.S. Pat. Publ. No. 20030113713,U.S. patent application Ser. No. 10/980,198, filed on Nov. 3, 2004,entitled “Modular Assay Plates, Reader System and Methods For TestMeasurements,” published as U.S. Pat. Publ. No. 20050142033; and U.S.patent application Ser. No. 10/744,726, filed on Dec. 23, 2003, entitled“Assay Cartridges and Methods of Using Same,” published as U.S. Pat.Publ. No. 20040189311, each of which is incorporated by this reference.One approach to multiplexing binding assays involves the use ofpatterned arrays of binding reagents (see, e.g., U.S. Pat. Nos.5,807,522 and 6,110,426, both entitled “Methods for FabricatingMicroarrays of Biological Samples” issued Sep. 15, 1998 and Aug. 29,2000 respectively, Delehanty J B, Printing functional proteinmicroarrays using piezoelectric capillaries, Methods Mol Biol. (2004)278:135-44; Lue R Y, Chen G Y, Zhu Q, Lesaicherre M L, Yao S Q,Site-specific immobilization of biotinylated proteins for proteinmicroarray analysis, Methods Mol Biol. (2004) 278:85-100; Lovett,Toxicogenomics: Toxicologists Brace for Genomics Revolution, Science(2000) 289: 536-537; Berns A., Cancer: Gene expression in diagnosis,Nature (2000) 403, 491-492; Walt, Molecular Biology: Bead-basedFiber-Optic Arrays, Science (2000) 287: 451-452 for more details).Another approach involves the use of binding reagents coated on beadsthat can be individually identified and interrogated. InternationalPatent publication WO9926067A1 (Watkins et al.) describes the use ofmagnetic particles that vary in size to assay multiple analytes;particles belonging to different distinct size ranges are used to assaydifferent analytes. The particles are designed to be distinguished andindividually interrogated by flow cytometry. Vignali has described amultiplex binding assay in which 64 different bead sets ofmicroparticles are employed, each having a uniform and distinctproportion of two dyes (Vignali, D. A. A., “Multiplexed Particle-BasedFlow Cytometric Assays,” J. Immunol. Meth. (2000) 243:243-255). Asimilar approach involving a set of 15 different beads of differing sizeand fluorescence has been disclosed as useful for simultaneous typing ofmultiple pneumococcal serotypes (Park, M. K. et al., “A Latex Bead-BasedFlow Cytometric Immunoassay Capable Of Simultaneous Typing Of MultiplePneumococcal Serotypes (Multibead Assay),” Clin Diagn Lab Immunol.(2000) 7:486-9). Bishop, J. E. et al. have described a multiplexsandwich assay for simultaneous quantification of six human cytokines(Bishop, J. E. et al., “Simultaneous Quantification of Six HumanCytokines in a Single Sample Using Microparticle-based Flow CytometricTechnology,” Clin Chem. (1999) 45:1693-1694).

Advantageously, in certain embodiments, tests may be conducted on asingle sample including, but not limited to, blood, serum, plasma, hair,sweat, urine, feces, tissue, biopsies, saliva, skin, mucosa, CNS fluid,bone marrow, tissue extracts, cells, cell extracts, cell culturesupernatants, and lymphatic fluids. Particularly advantageous are blood,blood serum, blood plasma, fecal matter, biopsy tissue, intestinalmucosa and urine. Specifically advantageous are blood, blood serum,blood plasma, fecal and urine samples due to the easy and non-surgicallyinvasive collection techniques.

A diagnostic test may also be is conducted in a single assay chamber,such as a single well of an assay plate or an assay chamber that is anassay chamber of a cartridge. The assay modules (for example assayplates or cartridges, or multi-well assay plates), methods andapparatuses for conducting assay measurements suitable for the presentinvention are described, for example, in U.S. patent application Ser.Nos. 10/185,274 and 10/185,363, both filed on Jun. 28, 2002, entitled“Assay Plates, Reader Systems and Methods For Luminescence TestMeasurements,” published as U.S. Pat. Publ. No. 20040022677 andUS20050052646, respectively, U.S. patent application Ser. No.10/980,198, filed on Nov. 3, 2004, entitled “Modular Assay Plates,Reader System and Methods For Test Measurements,” published as U.S. Pat.Publ. No. 20050142033, and U.S. patent application Ser. No. 10/744,726,filed on Dec. 23, 2003, entitled “Assay Cartridges and Methods of UsingSame,” published as U.S. Pat. Publ. No. 20040189311, each of which isincorporated by this reference. Assay plates and plate readers are nowcommercially available (MULTI-SPOT® and MULTI-ARRAY™ plates and SECTOR™instruments, Meso Scale Discovery, a division of Meso Scale Diagnostics,LLC, Gaithersburg, Md.).

Various diagnostic tests of the present invention may be furthersupplemented with a diagnostic test to determine if the patient hasviral or bacterial infection. Thus, in certain embodiments, theinvention further comprises determining if the patient has viral orbacterial infection. Various diagnostic tests of the present inventionmay be further supplemented with visual patient observation by thedoctor, radiological testing and/or histological testing of the patient.The methods of the invention may further comprise administering to thetested patient an effective amount of drug for effective treatment ofthe diagnosed IBD.

The function and advantage of these and other embodiments of the presentinvention may be more fully understood from the examples below. Thefollowing examples, while illustrative of certain embodiments of theinvention, do not exemplify the full scope of the invention.

EXAMPLES

The following examples are illustrative of some of the methods andinstrumentation falling within the scope of the present invention. Theyare, of course, not to be considered in any way limitative of theinvention. Numerous changes and modifications can be made with respectto the invention by one of ordinary skill in the art without undueexperimentation.

Materials & Methods:

Multi-Well Plates for Electrochemiluminescence Measurements:

Electrochemiluminescence measurements were carried out using multi-wellplates having integrated carbon ink electrodes (MULTI-SPOT® plates fromMeso Scale Discovery, a division of Meso Scale Diagnostics, LLC.). Adielectric layer patterned over the working electrode in each wellexposed ten regions or “spots” on the working electrode. The basicdetection technology is described in U.S. patent application Ser. Nos.10/185,274 and 10/185,363, both filed on Jun. 28, 2002, entitled “AssayPlates, Reader Systems and Methods For Luminescence Test Measurements,”published as U.S. Pat. Publ. No. 20040022677 and US20050052646,respectively. Kits for multiplexed measurements of cytokines using thistechnology are available from Meso Scale Diagnostics, LLC, Gaithersburg,Md. The kits include plates with an array of capture antibodies on the“spots” of each well. The measurements described herein used twodifferent cytokine kits that each measured a different 10 cytokinepanel. The first kit measured IL-1β, IL-12p70, IL-10, IL-2, GM-CSF,TNF-α, IL-8, IL-4, IL-5, and IL-6. The second kit measured Eotaxin,IFN-α, IFN-γ, sIL-6R, IL-12(total), IL-13, MCP-1, MIP-1β, RANTES, andsTNF-RII. The kits also included detection antibodies for each analyte(labeled with MSD Sulfo-TAG, an electrochemiluminescent label), an assaydiluent (MSD Serum Cytokine Diluent) and an antibody diluent (MSDCytokine Antibody Diluent). The labeled antibodies for each panel weremixed and diluted to a concentration of 2.5 ug/ml of each anti body inthe antibody diluent.

Sample Preparation

Matched human serum and EDTA plasma samples were obtained fromnon-diseased individuals and individuals with either Crohn's disease orulcerative colitis. The patients were diagnosed with either Crohn's orulcerative colitis according to current medical practice. Diseasedpatients were not undergoing therapy at the time of sample draw.Non-diseased (normal) individuals were defined as having normal generalappearance, height and weight, blood pressure, temperature and skin freeof lesions. A detailed questionnaire was also filled out by theseindividuals, and those displaying any evidence of any current diseasewere excluded from this normal group.

Samples were diluted 1:50 for the sIL-6R, RANTES, and sTNF-RIImeasurements. All other cytokines were measured using undiluted samples.

Calibrators

Calibrators containing known concentrations of all cytokines in a givenpanel were prepared in the assay diluent.

Electrochemiluminescence Measurement Instrument

Electrochemiluminescence was induced and measured in the MULTI-SPOT®plates using a SECTOR™ Imager 6000 (Meso Scale Discovery, a division ofMeso Scale Diagnostics, LLC., Gaithersburg, Md.).

Example 1: Cytokine Detection and Determination of Suitability ofCytokines to Act as Diagnostic Markers of IBD

Cytokine levels were measured in a multiplexed solid-phase sandwichimmunoassay.

The plates were blocked with blocking buffer (200 ul/well of Blocker A,MSD Discovery, a division of Meso Scale Diagnostics, LLC.) for 1 hour atroom temperature and then washed with PBS (3.times.250 ul). Assaydiluent (25 ul) was then added to each well and the plate incubated for30 minutes. Calibrators and serum/plasma samples were then added to theplates (25 ul/well). Each plate contained three replicates of eachcalibrator (8 levels) and three replicates of each sample. The plateswere then incubating with shaking for 2 hours. The mixture of detectionantibodies was then added to the plates (10 ul/well) and the plateincubated for another 2 hours with shaking. The plates were then washedwith PBS (3×250 ul), MSD Read Buffer T was added (150 ul/well) and theplates read on a Sector 6000 instrument. Cytokine concentration levelswere calculated by back-fitting to 4 parameter logistic fits to thecalibration curves for each analyte.

Table 2 lists, for each cytokine, one indicator of the utility of thecytokine as a marker for distinguishing between normal and diseasedpatients using ten patients clinically diagnosed with Crohn's disease,ten patients clinically diagnosed with ulcerative colitis and tenhealthy individuals. The utility is presented as a statisticallyweighted difference between control individuals and diseased patients,calculated as

$\frac{D - N}{\sqrt{\sigma_{D}*\sigma_{N}}}$

where D is the median concentration of a cytokine in patients diagnosedas having ulcerative colitis or Crohn's disease, N is the median of thecontrol individuals, σ_(D) in the standard deviation of D and σ_(N) isthe standard deviation of N. The larger the magnitude, the greater thestatistical difference between the diseased and normal populations. Themeasured levels for IL-1β, GM-CSF, IFN-α, IFN-γ and IL-13 were, for themost part, below the detection limits for these assays. The results areplotted in FIGS. 3-16.

TABLE 2 Analyte Crohn's UC IL-1β 0.4 1.8 IL-12p70 1.0 0.4 IL-10 −0.1−0.3 IL-2 1.1 0.5 TNF −1.5 −0.8 IL-8 0.7 0.0 IL-4 1.1 0.7 IL-5 0.4 0.3IL-6 0.4 1.1 Eotaxin −2.3 −2.4 IFN-γ 0.2 0.7 sIL-6R −1.6 −1.4IL-12(total) −0.1 −0.5 M IP- 1β −3.7 −5.1 MCP-1 −2.3 −3.6 RANTES −2.6−1.7 sTNFRII 1.5 5.0

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims.

The present invention is directed to each individual feature, system,material and/or method described herein. In addition, any combination oftwo or more such features, systems, materials and/or methods, providedthat such features, systems, materials and/or methods are not mutuallyinconsistent, is included within the scope of the present invention.

In the claims (as well as in the specification above), all transitionalphrases or phrases of inclusion, such as “comprising,” “including,”“carrying,” “having,” “containing,” “composed of,” “made of,” “formedof,” “involving” and the like shall be interpreted to be open-ended,i.e. to mean “including but not limited to” and, therefore, encompassingthe items listed thereafter and equivalents thereof as well asadditional items. Only the transitional phrases or phrases of inclusion“consisting of” and to “consisting essentially of” are to be interpretedas closed or semi-closed phrases, respectively. The indefinite articles“a” and “an,” as used herein in the specification and in the claims,unless clearly indicated to the contrary, should be understood to mean“at least one.”

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties. In cases where thepresent specification and a document incorporated by reference and/orreferred to herein include conflicting disclosure, and/or inconsistentuse of terminology, and/or the incorporated/referenced documents use ordefine terms differently than they are used or defined in the presentspecification, the present specification shall control.

1.-18. (canceled)
 19. A multiplexed assay kit used to evaluate theefficacy of a treatment regimen in a patient diagnosed with inflammatorybowel disease, wherein said kit comprises an assay cartridge forconducting a plurality of assays, wherein said cartridge is configuredto measure a level of a plurality of biomarkers in a patient sample,said plurality of biomarkers comprises soluble TNF receptor II (sTNFRII)and MIP-1beta, and at least one additional biomarker comprising eotaxin,sIL-6R, MCP-1, RANTES, and combinations thereof.
 20. The kit of claim 19wherein the multi-well assay plate is capable of communicating with aprocessor, the processor configured to determine a ratio of sTNFRII toMIP-1 beta in samples from said patients versus people not diagnosedwith inflammatory bowel disease.
 21. The kit of claim 19, furthercomprising in one or more vials, containers, or compartments, a set oflabeled detection antibodies specific for said plurality of biomarkers.22. The kit of claim 19, wherein said kit is further configured tocompare said level to a level of a normal control.
 23. The kit of claim19, wherein said at least one additional biomarker comprises MCP-1. 24.The kit of claim 19, wherein said kit is configured to measure saidlevel using an immunoassay.
 25. The kit of claim 19, wherein saidcartridge comprises a flow cell having an inlet, an outlet or adetection chamber, said inlet, detecting chamber, or outlet defining aflow path through said flow cell, said detection chamber configured tomeasure said level of said plurality of biomarkers and said at least oneadditional biomarker in said sample.
 26. The kit of claim 19, whereinsaid kit further comprises one or more additional assay reagents used insaid assay, said one or more additional assay reagents provided in oneor more vials, containers, or compartments of said kit.
 27. The kit ofclaim 21, further comprising, in said one or more vials, containers, orcompartments, a set of calibrator proteins.
 28. The kit of claim 19,further comprising one or more diluents.
 29. The kit of claim 21,wherein said detection antibodies are labeled with anelectrochemiluminescent (ECL) label.
 30. The kit of claim 19, whereinsaid kit further comprises an ECL read buffer.
 31. The kit of claim 19,further comprising capture antibodies to at least two additionalbiomarkers selected from the group consisting of eotaxin, sIL-6R, MCP-1,and RANTES.
 32. The kit of claim 20, wherein the processor is furtherconfigured to detect a ratio of sTNFRII to sIL-6R in samples from saidpatients versus people not diagnosed with inflammatory bowel disease.33. The kit of claim 20, wherein the processor is further configured todetect a ratio of MIP-1beta to MCP-1 in samples from said patientsversus people not diagnosed with inflammatory bowel disease.
 34. The kitof claim 20, wherein the processor is further configured to detect aratio of RANTES to MCP-1 in samples from said patients versus people notdiagnosed with inflammatory bowel disease.
 35. The kit of claim 19,wherein the samples from said patients are selected from the groupconsisting of blood, serum, plasma, fecal, urine and combinationsthereof.